(1) Field of the Invention
This invention relates to a novel mutant strain of Listeria monocytogenes, to its use in the production of high titers of IgM antibodies specific thereto, to its use and functioning as an immunopotentiating agent (or immunostimulant adjuvant) when conjugated to a sensitizing antigen, to the antibodies responsive to the mutant strain or conjugate, and to such conjugates used as immunotherapeutic agents for use in the treatment of various diseases.
(2) Description of the Prior Art
Immunity is an everyday word applied to a special category of defenses possessed by the body, i.e., the reticuloendothelial and lymphoproliferation systems, by means of which infectious agents, e.g., antigens, may be checked or destroyed even after they have entered the body tissues. The immunity is largely due to the development within the body of substances known as antibodies (or immune bodies) which interact specifically with the antigen destroying or inactivating the disease causing agent. Immunity may be natural or acquired, and in the latter case may be acquired naturally or artificially. Artificial immunity, as is well known, can be either passive, i.e., by injection of an antiserum (prophylactic, therapeutic), or active, as by vaccination with, for example, live or dead organisms.
Antigens, which may either be introduced into or formed in the body, generally speaking may be defined as those physiological substances such as proteins, animal products, bacteria, viruses, hormones, chemical vitamins, lipopolysaccharides, peptides, etc., which, when injected into an animal system, human or not, stimulate the production of a specific protein mechanism, i.e., the antibody response, which enables the animal injected to defend or react against that antigen. A further, more detailed, description of antigens is set forth in P. L. Carpenter, Immunology and Serology, (2d Edition, 1968); however, typical antigens may be classified as follows:
(1) protein antigens, such as ceruloplasmin and serum albumin; PA1 (2) bacterial antigens, such as teickoic acids, flagellar antigens, capsular polysaccharides, and extra-cellular bacterial products and toxins; PA1 (3) blood group antigens, such as glycoproteins and glycolipids; PA1 (4) viruses, such as animal, plant, and bacterial viruses; PA1 (5) conjugated and synthetic antigens, such as proteinhapten conjugates, and synthetic polypeptides; and PA1 (6) nucleic acides, such as ribonucleic acid and deoxyribonucleic acid.
By the term antibody is meant broadly any substance which is mutually attracted to the antigen and which can be observed to combine with and have a neutralizing effect on the antigen. Antibodies, from a somewhat more specific definition, are those physiological agents which, in an animal system, stimulate the production of a serum protein called immunoglobulins (or sometimes "gamma globulins") for defending against the antigen encountered. The immunoglobulins (Ig) are actually a family of closely related though not identical proteins, of which there are five major types, IgG, IgA, IgD, IgE, and IgM, varying in molecular weight from approximately 160,000 to approximately 1,000,000. The IgG (or immunoglobulin gamma G) type is the most prevalent "antibody" in a serum and is characterized by a molecular weight of about 160,000. The IgM type is the least prevalent and is characterized by a molecular weight of about 1,000,000.
Although the antigen-antibody reaction forms the basis for immunity, that is the state of being immune to or protected from disease, especially an infectious disease, the reaction also is the basis for the whole field of immunochemistry which field includes not only immunization but also diagnostic medicine and immunotherapy.
Immunization procedures are important in the prevention of various diseases including viral diseases. Sometimes it is necessary that a large supply of antibodies appear in a person's blood immediately in order to combat an overwhelming infection already present in the body. Accordingly, the patient must receive ready-made antibodies, and various commercial means of manufacture and recovery of various antibodies have been developed over the years.
In more recent years, there has grown an ever increasing need for commercial sources of antibodies for use in various immunoassays, both radioimmunoassays and, even more recently, enzymeimmunoassays. Such assays are used for various purposes, e.g., determination of pregnancy, detecting the presence of heroin and other opiate alkaloids in a person's physiological fluids, e.g., urine, and detection of hepatitis.
There has also developed considerable evidence that viruses cause various kinds of tumors and cancerous growths, particularly in lower animals such as rabbits, mice, chickens, and hamsters. Various investigators, including the present Applicant, have also been active in their attempt to discover agents that might be effective in cancer immunotherapy. See Likhite, V. V., "Clinical Cancer Immunotherapy: Experience in Breast and Lung Cancer", in Immunocancerology in Solid Tumors, (Martin, M. and Dionne, L., eds.) pp. 135-141. (Stratton, 1976). "Rejection of Tumor Metastases in Fisher 344 Rats Following Administration of Killed Corynebacterium parvum", Cancer Immunology and Immunotherapy, (1977), Vol. 2, pp. 173-178 V. V. Likhite. Thus, with this and other interests in immunotherapy there is a continuing and growing need for antibodies.
Basically, there are two methods for the isolation and purification of antibodies. One method involves separation of groups of molecules based upon their physical properties such as molecular weight. Inherent in this approach is the fact that similar molecules regardless of immunological specificity will be isolated together. The second method of isolation of antibodies involves immunological methods which depend upon a primary characteristic of all antibodies, i.e., their ability to react with specific antigens. Thus, if an antigen is added to a serum containing a specific antibody, the antigen and antibody will complex and precipitate from the solution: EQU Antigen+Antibody=Antigen-Antibody Complex
Once separated from the solution the antigen can be removed from the complex by dissociation followed by physical separation if the two molecules are sufficiently different. The basic difficulty with the above separation technique is that, in general, complete separation of antigen from antibody is not achieved. As a result, there is always some residual antigen left in any antibody recovered, making for an antibody preparation of less than the desired specificity.
Campbell et al., Proc. Nat'l. Acad. Sci. (U.S.) 37,575 (1951) discovered that antibodies could be isolated and purified which did not contain residual antigen by covalently coupling antigens to insoluble polymers before reacting with antibody in a serum sample. Since these earlier studies on immuno-adsorbents, several types of polymers have been employed as carriers for antigens. These include cellulose and its derivatives, polyamino-polystyrene, dextrose, and polyaminoacids. Generally, these materials have been found satisfactory; however, each has certain specific drawbacks. Some of the problems with these materials involve (a) release of nonspecific protein adsorbed onto the carrier earlier from serum with release of the antibody; (b) decreasing flow-rates from the immunoadsorbent column; (c) immunoadsorbent efficiency, i.e., the percentage of antibody retained on the immunoadsorbent in relation to the total antibody added; (d) the period of usefulness of the particular immunoadsorbent and yield, due to irreversible complexing resulting in fewer sites available for reuse; and (e) loss of biological activity. As a result, the search has continued over the years, and still continues, for improved methods for isolating and purifying antibodies.
Examples of this continuing search are the inventions disclosed in U.S. Pat. Nos. 3,652,761 and 3,843,444. In the former patent, either antigens or antibodies are disclosed to be stabilized by chemical coupling to an inorganic carrier by means of an intermediate silane coupling agent rather than being merely adsorbed onto a carrier. These immunochemical composites, according to the patentee, are biologically active, have acceptable capacity, excellent antigen-antibody association-dissociation characteristics, and can be reused over and over many times. Nevertheless, the fact remains that this procedure of isolation of antibodies from antigens is attendant with some of the very same problems as earlier procedures where, e.g., an antigen was insolubilized on an immunoadsorbent. This results from the mutual attraction that antibodies and antigens have for one another.
The approach disclosed in U.S. Pat. No. 3,843,444, which issued to the Applicant herein on Oct. 22, 1974, for separating biological substances such as antibodies and antigens from a liquid is to make use of the mutual attraction that such substances have for one another. However, the antibodies and antigens are prevented from actually contacting one another by the interdisposition of a thin semipermeable membrane, to the opposite sides of which the antibodies and antigens are attracted. The antibodies are then subsequently washed from the membrane surface by hypertonic saline at 37.degree. C., after the membrane surface is first washed with physiological saline, to wash away the non-antibody protein attracted to the surface. Thus, a rather high percentage of the available antibody is recovered in a rather simple process and with substantially unimpaired physiological properties.
Nevertheless, even with ever improving methods of isolation and purification of antibodies, the fact remains that the availability of antibodies for immunization, diagnostic or therapeutic purposes is still limited due to the limit of conferred antibody response in the host. Moreover, when an animal receives repeated injections of a given antigen, the induced specific antibody response in the host animal against the injected antigen represents a relatively small amount, usually less than 1% of the serum globulin pool.
Thus, there is still a need not only for a method of producing larger quantities of antibodies within a biological system, but also improved methods of recovering antibodies from serum and without appreciable loss of activity.
Heretofore others have disclosed the adjuvant effect with the combined immunization with two or more antigens in a single injection. Each antigen is reinforced and simultaneously produces immunity. Moreover, Meacock et al., Chemical Abstracts, 79 64410X (1973) Effect of adjuvant and stress in the production of IgE antibody in rats, disclose the enhanced production of IgE antibody in rats through injection of Bordetella pertussis cells within twenty-four hours before or after the injection of antigen. Likhite, V. V., "Clinical Cancer Immunotherapy: Experience in Breast and Lung Cancer", Immunocancerology in Solid Tumors, supra, discloses that killed Cornebacterium parvum had been discovered by others earlier not only to be effective as an immunostimulant adjuvant but also to have immunotherapeutic properties toward neoplasms. Thus, Likhite, as disclosed in that publication, investigated further the possible immunotherapeutic effects of B. pertussis, widely confirmed to have immunopotentiating properties, toward cancer. The results revealed that only the group of animals treated with a cancer cell (e.g., T1699) - B. pertussis conjugate survived. The conjugate was formed by coupling the tumor cells at 4.degree. C. to toluene-2,4-diisocyanate followed by coupling the killed B. pertussis microorganism to the 2d carbon position at 37.degree. C.
Numerous other investigators have been active in recent years in their attempt to discover agents that might be effective in cancer immunotherapy. British Pat. No. 1,193,378 discloses a therapeutic agent which comprises water-soluble cancer antigens such as human tumor or leukemia antigen coupled to foreign proteins such as gamma globulins used to vaccinate human patients against cancer. When such an antigen complex is used, as disclosed in that patent, the therapeutic effect is to retard or at least slow down the progress of the disease involved in the patient. The patentee hypothesized that the entire complex acts as an antigen causing the patient to manufacture antibodies in his blood against the vaccine complex and each of its components, soluble human cancer antigens as well as the foreign protein portion of the complex. According to the patentee anti-human tumor and anti-rabbit gamma globulin anti-bodies may be demonstrated in the vaccinated patient's sera by titering their sera against the vaccine complex and each of its components (soluble human cancer antigen and soluble rabbit gamma globulin). The antibodies thus developed, the patent discloses, apparently attack the cancer antigens in the patient such as tumor antigens and the tumor itself and cause the rate of growth to slow down or even cease.